Home › Microbiology › A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
Microbiology JoVE (Open Access) Citable · DOI

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

DOI: 10.3791/4316-v
What you'll learn
  • ✓ Isolate platelet-rich and platelet-poor plasma from clinical blood samples
  • ✓ Prepare Plasmodium falciparum-infected erythrocytes for clumping assays
  • ✓ Perform platelet-mediated clumping assay using Plasmagel flotation
  • ✓ Quantify and interpret platelet-dependent rosetting phenotypes in resource-limited settings
Protocol

Biopharma Insights This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Difficulty
intermediate
Total time
~4–6 hours (includes plasma preparation, parasite culture timing, and assay execution)
Model organism
Plasmodium falciparum clinical isolates; human erythrocytes and platelets
Biosafety
BSL-2

Steps

1
Prepare platelet-rich and platelet-poor plasma

Centrifuge whole blood at low speed to obtain platelet-rich plasma (PRP), then further centrifuge PRP at high speed to generate platelet-poor plasma (PPP) as a control.

▶ 01:20
2
Culture Plasmodium falciparum parasites

Maintain P. falciparum clinical isolates in standard culture conditions to generate sufficient infected erythrocytes for the assay.

▶ 02:17
3
Prepare thin blood film slides

Create thin blood films from cultured parasite suspensions and stain to verify parasitemia levels and morphology.

▶ 02:58
4
Perform Plasmagel flotation enrichment

Use Plasmagel density-based separation to enrich mature infected erythrocytes and remove uninfected cells from the parasite suspension.

▶ 03:57
5
Execute platelet-mediated clumping assay

Co-incubate platelet-rich plasma with enriched infected erythrocytes, then quantify clump formation as a measure of platelet-dependent rosetting phenotype.

▶ 05:01
6
Analyze and interpret clumping results

Examine prepared slides under microscopy, count clumped versus single infected erythrocytes, and compare PRP versus PPP conditions to determine platelet-mediated adhesion.

▶ 06:08
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